Thesis Effects of dietary supplementation with selected plant extracts on the physiological parameters of striped catfish (pangasianodon hypophthalmus) doctoral dissertation major: Aquaculture (9620301) 2023

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY PHAM NGOC NHU EFFECTS OF DIETARY SUPPLEMENTATION WITH SELECTED PLANT EXTRACTS ON THE PHYSIOLOGICAL PARAMETERS OF STRIPED CATFISH (Pangasianodon hypophthalmus) DOCTORAL DISSERTATION MAJOR: AQUACULTURE (9620301) 2023 MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY PHAM NGOC NHU Ph.D. student ID: P0618001 EFFECTS OF DIETARY SUPPLEMENTATION WITH SELECTED PLANT EXTRACTS ON THE PHYSIOLOGICAL PARAMETERS OF STRIPED CATFISH (Pangasianodon hypophthalmus) DOCTORAL DISSERTATION MAJOR: AQUACULTURE (9620301) SUPERVISOR Prof. Dr. Do Thi Thanh Huong Assoc. Prof. Dr. Bui Thi Bich Hang 2023 DATA SHEET Title: Effects of dietary supplementation with selected plant extracts on the physiological parameters of striped catfish (Pangasianodon hypophthalmus) Subtitle: Ph.D. Dissertation Author: Pham Ngoc Nhu, Ph.D. student ID: P0618001 Affiliation: Faculty of Aquatic Biology and Environmental Science, College of Aquaculture and Fisheries, Can Tho University, Vietnam Publication year 2023 Cited as: Nhu, Pham Ngoc (2023). Effects of dietary supplementation with selected plant extracts on the physiological parameters of striped catfish (Pangasianodon hypophthalmus). Doctoral Dissertation. College of Aquaculture and Fisheries, Can Tho University, Vietnam. Keywords: Digestive enzyme activities, hematological parameters, growth, oxidative stress, Pangasianodon hypophthalmus, plant extracts Supervisors: Prof. Dr. Do Thi Thanh Huong, College of Aquaculture and Fisheries, Can Tho University, Viet Nam. Assoc. Prof. Dr. Bui Thi Bich Hang, College of Aquaculture and Fisheries, Can Tho University, Viet Nam. i RESULT COMMITMENT I certify that this dissertation was thoroughly researched using all of the findings of my research. The data and results presented in the dissertation were believable and had never been published. These data and results are entirely applicable to the BioAquaActive project. Can Tho, .., 2023 Supervisors Ph.D. Student Prof. Dr. Do Thi Thanh Huong Pham Ngoc Nhu Assoc. Prof. Dr. Bui Thi Bich Hang ii ACKNOWLEDGEMENTS First of all, I would like to express my sincere appreciation to Prof. Dr. Do Thi Thanh Huong, Assoc. Prof. Dr. Bui Thi Bich Hang and Prof. Dr. Nguyen Thanh Phuong for offering me the opportunity to properly study the Ph.D. program. These teachers enthusiastically accompanied and addressed the concepts, reviewed the dissertation details, and provided me with encouragement throughout the study; and have always created the best conditions for me to accomplish the research thoroughly, not only during the study but also when I experienced problems or failures. From the smallest attention to the valuable experiences that the professors have taught, it has always been a source of encouragement to promote motivation. The fact that I am currently completing the dissertation and the source of the gathered knowledge is proof of the accomplishment of my efforts. I would like to thank Prof. Dr. Patrick Kestemont for his encouragement, motivation, and assistance in accomplishing my responsibilities, not just in terms of knowledge but also in my experiences at Namur University, Belgium. The far distance from home, family, relatives, and friends, as well as the experience of visiting a foreign country in terms of culture and language (I initially assumed that Belgian citizens spoke English rather than French), did not prevent me from being concerned. On the other hand, Professor Patrick acknowledged all of my anxieties and always created the most supportive conditions for me to rapidly integrate and approach the learning experience and research appropriately. I'd like to thank Patrick's team members (Robert, Emily, Enoha, Hossain, Mai,...) for their assistance in improving my analytical skills, as well as the full support of equipment and materials well as the retention of great experiences at Namur University. Unfortunately, the Covid-19 pandemic outbreak while I was experiencing in Belgium, and my studies seemed abrupt. Everything would probably be stuck if it weren't for the professor's encouragement and assistance in completing my responsibilities completely. Once again, I would like to thank Professor Patrick and the members of the team for accompanying me in my beloved Belgium. I’d like to thank my companions Dang Quang Hieu, Nguyen Tinh Em, and especially Ms. Nguyen Thi Kim Ha for their experience in laboratory analysis techniques, chemicals, and materials at Can Tho University (CTU), as well as, their accompanying each other during the above experience at Namur University. I would want to express my sincere appreciation to Dr. Do Van Buoc for granting me the opportunity to access and participate in the AquaBioactive project, as well as to complete this dissertation. Being with him for the initial two years of my study was a pleasure and an unforgettable experience, as well as an advisor who assisted me when I encountered my initial difficulties. Furthermore, my heartfelt gratitude to Ms. Hong Mong Huyen, who always promotes the spirit, relieves stress, and shares sorrows and iii pleasures, as well as experiences, during the period in Belgium, as well as at CTU's dormitory. In addition, I would like to thank the physiology team at the College of Aquaculture and Fisheries (CAF), Can Tho University for their great assistance starting from the first day of implementing my study. I'd like to thank Ms. Le Thi Bach, who helped with the supply of plant extracts for research, as well as the staff of CAF as Assoc. Prof. Dr. Tran Minh Phu and Dr. Nguyen Le Anh Dao, aided me with experimental materials for my experiments. I also appreciate the collaborative efforts of four female masters (Nguyen Thi Hoi, Tran Thi Phuong Hang, Vien Tuyen Anh, and Doan Anh Thu) who have accompanied together throughout my study; as well as the students in the Faculty of Aquatic Biology and Environmental Science, who have supported and shared the happiness and assistances to implement my experiments. Finally, I'd want to express my gratitude to my family and friends for always loving and supporting me during my studies. iv TABLE OF CONTENTS DATA SHEET................................................................................................................. i RESULT COMMITMENT ......................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................ iii LIST OF FIGURES ...................................................................................................... ix LIST OF TABLES ........................................................................................................ xi LIST OF ABBREVIATION ..................................................................................... xiii ABSTRACT ................................................................................................................ xiv TÓM TẮT ................................................................................................................... xvi CHAPTER 1 ................................................................................................................. 18 INTRODUCTION ....................................................................................................... 18 1.1 General introduction ................................................................................................ 18 1.2 The objectives of the dissertation ............................ 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Bookmark not defined. 1.3 The main contents of the dissertation ...................................................................... 20 1.4 The hypotheses of the dissertation .......................................................................... 21 1.5 New findings of the dissertation .............................................................................. 21 1.6 Significant contributions of the dissertation ............................................................ 22 CHAPTER 2 ................................................................................................................. 22 LITERATURE REVIEW ........................................................................................... 23 2.1 The status and importance of aquaculture and fisheries.......................................... 23 2.2 Climate changes and impacts on aquaculture and fisheries .................................... 27 2.3 Mechanism of stress on fish .................................................................................... 28 2.4 Effects of environmental factors on fish ................................................................. 30 2.4.1 Effect of temperature on fish ................................................................................ 30 2.4.2 Effects of salinity on fish ...................................................................................... 37 2.4.3 Effects of nitrite on fish ........................................................................................ 43 2.5 Effects of plant extracts on fish ............................................................................... 47 2.5.1 Effects of plant extracts on hematological parameters of fish ............................. 47 2.5.2 Effects of plant extracts on digestive enzymes activities and growth of fish ...... 49 2.5.3 The effects of plant extracts on oxidative stress of fishes .................................... 60 2.5.4 Effects of plant extracts on striped catfish (P. hypophthalmus) ........................... 62 a) Effects of Mimosa pudica on aquaculture species .................................................... 67 b) Effects of Psidium guajava on aquaculture species .................................................. 67 c) Effect of Phyllanthus amarus on fish ........................................................................ 71 d) Effect of Euphorbia hirta on fish ............................................................................... 77 e) Effect of Azadirachta indica on fish .......................................................................... 79 CHAPTER 3 ................................................................................................................. 81 EFFECTS OF PLANT EXTRACTS ON SELECTED HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES, AND GROWTH PERFORMANCE v OF STRIPED CATFISH, Pangasianodon hypophthalmus (Sauvage, 1878) FINGERLINGS ........................................................................................................... 81 Abstract ........................................................................................................................ 81 3.1 Introduction ............................................................................................................. 81 3.2 Material and Method ............................................................................................... 83 3.2.1 Plant extract and feed preparation ........................................................................ 83 3.2.2 Experimental fish acclimation, facilities, and feeding management .................... 84 3.2.3 Haematological and biochemical parameters ....................................................... 85 3.2.4 Digestive enzyme activities .................................................................................. 86 3.2.5 Growth performance and survival rate ................................................................. 86 3.2.6 Statistical analysis ................................................................................................ 87 3.3 Results ..................................................................................................................... 87 3.3.1 Effects of plant extracts on haematological parameters ....................................... 87 3.3.2 Effects of plant extract on digestive enzyme activities ........................................ 90 3.3.3 Effects of plant extracts on growth performance and the survival rate ................ 91 3.4 Discussion ................................................................................................................ 92 3.5 Conclusions ............................................................................................................. 96 CHAPTER 4 ................................................................................................................. 97 EFFECTS OF Psidium guajava AND Phyllanthus amarus EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO HIGH-TEMPERATURE STRESS ........................................................................................................................ 97 4.1. Introduction ............................................................................................................ 98 4.2. Materials and methods .......................................................................................... 100 4.2.1 Plant extracts and diet preparation ..................................................................... 100 4.2.2 Experimental fish acclimation, facilities, and feeding trial ................................ 101 4.2.3 Haematological and biochemical parameters ..................................................... 102 4.2.4 Oxidative stress assays ....................................................................................... 103 4.2.5 Statistical analysis .............................................................................................. 104 4.3. Results .................................................................................................................. 105 4.3.1 Effect of plant extract on P. hypophthalmus haematological parameters .......... 105 4.3.2 Effect of plant extract on digestive enzyme activities ........................................ 109 4.3.3 Effect of plant extract on growth performance of P. hypophthalmus ................ 111 4.3.4 Effect of plant extract on P. hypophthalmus oxidative stress ............................ 113 4.4. Discussion ............................................................................................................. 115 4.5 Conclusions ........................................................................................................... 122 Acknowledgments ....................................................................................................... 122 CHAPTER 5 ............................................................................................................... 123 vi EFFECTS OF GUAVA (Psidium guajava L.) AND BHUMI AMLA (Phyllanthus amarus Chum et Thonn.) EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO SUBLETHAL SALINITIES ............................. 123 Abstract ...................................................................................................................... 123 5.1 Introduction ........................................................................................................... 123 5.2 Material and Method ............................................................................................. 125 5.2.1 Plant extract and feed preparation ...................................................................... 125 5.2.2 Experimental fish acclimation, facilities, and feeding management .................. 126 5.2.3 Haematological and biochemical parameters ..................................................... 127 5.2.4 Digestive enzyme activities ................................................................................ 127 5.2.5 Oxidative stress biomarkers ............................................................................... 128 5.2.6 Growth performance and survival rate ............................................................... 129 5.2.7 Statistical analysis .............................................................................................. 129 5.3 Results ................................................................................................................... 129 5.3.1 Effects of plant extracts on haematological parameters ..................................... 129 5.3.2 Effects of plant extracts on digestive enzyme activities..................................... 133 5.3.3 Effects of plant extracts on oxidative stress ....................................................... 136 5.3.4 Effects of plant extracts on growth performance and the survival rate .............. 140 5.4 Discussion .............................................................................................................. 140 5.5 Conclusions ........................................................................................................... 144 CHAPTER 6 ............................................................................................................... 126 EFFECTS OF Psidium guajava AND Phyllanthus amarus EXTRACTS ON HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES ACTIVITIES OXIDATIVE STRESS, AND GROWTH OF STRIPED CATFISH, Pangasianodon hypophthalmus FINGERLINGS EXPOSED TO NITRITE-INDUCED TOXICITY ..................................................................................................................................... 126 Abstract ........................................................................................................................ 126 6.1 Introduction ........................................................................................................... 126 6.2 Material and Method ............................................................................................. 128 6.2.1 Plant extract and feed preparation ...................................................................... 128 6.2.2 Experimental fish acclimation, facilities, and feeding management .................. 128 6.2.3 Haematological and biochemical parameters ..................................................... 130 6.2.4 Digestive enzyme activities ................................................................................ 130 6.2.5 Oxidative stress biomarkers ............................................................................... 131 6.2.6 Growth performance and survival rate ............................................................... 132 6.2.7 Statistical analysis .............................................................................................. 132 6.3 Results ................................................................................................................... 132 6.3.1 Effects of plant extracts on haematological parameters ..................................... 132 vii 6.3.2 Effects of plant extracts on digestive enzyme activities..................................... 136 6.3.3 Effects of plant extracts on oxidative stress ....................................................... 136 6.3.4 Effects of plant extracts on growth performance and the survival rate .............. 142 6.4 Discussion .............................................................................................................. 142 6.5 Conclusions ........................................................................................................... 146 CHAPTER 7 ............................................................................................................... 147 GENERAL DISCUSSION ........................................................................................ 147 7.1 Effects of five selected extracts on physiology parameters and stress mitigation of P. hypophthalmus fingerlings ...................................................................................... 147 7.2 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P. hypophthalmus fingerlings exposed to high- temperature stress ........................................................................................................ 150 7.3 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P. hypophthalmus fingerlings exposed to sublethal salinities ....................................................................................................................... 152 7.4 Effects of Psidium guajava and Phyllanthus amarus extracts on physiology parameters and stress mitigation of P. hypophthalmus fingerlings exposed to nitrite- induced toxicity ........................................................................................................... 153 CONCLUSIONS AND RECOMMENDATIONS .................................................. 156 8.1 Conclusion ............................................................................................................. 156 8.2 Recommendations for further studies .................................................................... 157 APPENDIX ..................................................................................................................... 1 List of protocols of analysis used in the studies .............................................................. 1 viii LIST OF FIGURES Figure 2.1 Total fisheries and aquaculture production 2020 (FAO, 2022). .................. 23 Figure 2.2 World fisheries and aquaculture production, utilization and trade .............. 24 Figure 2.3 World production of striped catfish (thousand tons) (FAO, 2022). ............. 24 Figure 2.4 Farming area and production of striped catfish in Mekong Delta (2015-2021; VASEP, 2022) .............................................................................................. 24 Figure 2.5 Export value of striped catfish in the period 2015-2021 (VASEP, 2022). .. 26 Figure 2.6. Stress response in response to temperature elevation.. .............................. 30 Figure 2.7 E. hirta L. (Igwe et al., 2016); Phyllanthus amarus (Abeng, 2017); Mimosa pudica (Goyal, 2014); Psidium guajava (Deepa et al., 2017); Azadirachta indica (Neelakantan et al., 2011). ................................................................ 66 Figure 2.8 Antioxidant activities of different A. indica extracts and their bioactive compounds. .................................................................................................. 80 Figure 3.1 Plasma glucose concentrations (mg/100 mL) of P. hypophthalmus fed extract-based diets in a 60-day experiment. ................................................. 87 Figure 3.2 The pepsin (A) and gastric amylase (B) activities (U min/mg protein) of P. hypophthalmus fed extract-based diets in a 60-day experiment. ..... 90 Figure 3.3 The intestinal amylase (A), trypsin (B) and chymotrypsin (C) activities (U min/mg protein) of P. hypophthalmus fed extract-based diets in a 60-day experiment.. .................................................................................................. 92 Figure 4.1 Pepsin (A) and gastric amylase (B) activities of P. hypophthalmus fingerlings under various temperatures for 42 days. .................................................... 110 Figure 4.2 Intestinal amylase (A), trypsin (B), and chymotrypsin (C) activities of P. hypophthalmus fingerlings under various temperatures for 42 days.. ....... 111 Figure 4.4 LPO-gill (A) and LPO-liver (B) of P. hypophthalmus fingerlings under various temperatures in 42 days. ............................................................... 112 Figure 4.3 Growth performance (WG, DWG, and SGR) and survival rates of P. hypophthalmus fingerlings under various temperatures for 42 days. .................................................................................................................... 112 Figure 4.5 LPO-brain (A) and LPO-muscle (B) of P. hypophthalmus fingerlings under various temperatures in 42 days. ...................................................... 113 Figure 4.7 CAT-gill (A) and CAT-liver (B) of P. hypophthalmus fingerlings under various temperatures in 42 days.. ............................................................... 115 Figure 5.1 RBCs (A), Hb (B), and Hct (C) of P. hypophthalmus fingerlings under various salinity in 14 days. Asterisk (*) present significant differences (p<0.05) among salinity levels whereas different lowercase letters (a, b) indicate significant differences among feed groups (p<0.05). ................................ 130 Figure 5.2 MCV (A), MCH (B), and MCHC (C) of P. hypophthalmus fingerlings under various ........................................................................................................ 131 ix Figure 5. 3 Glucose concentration (A), osmolality (B) of P. hypophthalmus fingerlings under various salinity in 14 days. Asterisk (*) presents significant differences (p<0.05) among salinity levels ................................................................... 132 Figure 5.4 LPO- liver (A), LPO-gill (B), LPO-muscle (C) and LPO-brain (D) of P. hypophthalmus fingerlings under various salinity in 42 days. Asterisk (*) present significant differences (p<0.05) among salinity levels whereas different lowercase letters (a, b) indicate significant differences among feed groups (p<0.05). ......................................................................................... 137 Figure 5. 5 CAT-liver (A), CAT-gill (B), CAT- brain (C) and CAT- muscle (D) of P. hypophthalmus fingerlings under various salinity in 42 days. Asterisk (*) present significant differences (p<0.05) among salinity levels whereas different lowercase letters (a, b) indicate significant differences among feed groups (p<0.05). ......................................................................................... 139 Figure 6.1 RBCs, Hb, and Hct of P. hypophthalmus fingerlings exposed to different nitrite concentrations for 14 days. .............................................................. 134 Figure 6.2 MCV (A), MCH (B), and MCHC (C) of P. hypophthalmus fingerlings exposed to different nitrite concentrations for 14 days. ............................. 135 Figure 6.3 Pepsin and gastric amylase activities of P. hypophthalmus fingerlings exposed to different nitrite concentrations for 42 days. ............................. 136 Figure 6.4 Intestinal amylase, trypsin, and chymotrypsin activities of P. hypophthalmus fingerlings exposed to different nitrite concentrations for 42 days.. ......... 137 Figure 6.5 DWG, WG, and survival rate of P. hypophthalmus fingerlings exposed to different nitrite concentrations for 42 days. ............................................... 142 x LIST OF TABLES Table 2.1 Effects of salinity on fish’s physiology under culture ................................. 41 Table 2.2 Effects of some herbal extracts on hematological parameters of fish under culture ................................................................................................ 51 Table 2.3 Effects of some herbal extracts on digestive enzyme activities of fish under culture ................................................................................................ 54 Table 2.4 Main oxygen reactive species and their performance Garcez et al. (2004) . 56 Table 2.5 Antioxidant defense system of cell enzymes and biological mechanism (Shi and Noguchi, (2001). ............................................................................ 56 Table 2.6 Main groups of plant compounds with antimicrobial activity (Cowan, 1999) .............................................................................................. 61 Table 2.7 Effects of some herbal extracts on oxidative stress indices of fish under culture ................................................................................................ 64 Table 2.8 Lists of plant extract (from World Flora Online, ............................................................... 65 Table 2.9 Identification information of five plant extracts used in the experiment (Dao et al., 2020). ......................................................................................... 66 Table 2.10 Phytochemical screening of Mimosa pudica ............................................... 68 Table 2.11 Bioactive metabolites of Mimosa pudica (Rizwan et al., 2022) ................. 68 Table 2.12 Phytochemical screening of Psidium guajava ........................................... 70 Table 2. 13 Phytochemical screening of different species of Psidium guajava ............ 72 Table 2.14 Bioactive metabolites of Psidium guajava .................................................. 72 Table 2.15 Bioactive metabolites of P. amarus (Patel et al., 2011) ............................. 76 Table 2.16 Phytochemical screening of Euphorbia hirta (Asha et al., 2014) ............... 78 Table 3.1 Experimental feed ingredients and formulation ........................................... 84 Table 3.2 Haematological parameters of P. hypophthalmus fed extract-based diets in a 60-day experiment ........................................................................................ 89 Table 3.3 Growth performance of P. hypophthalmus fed plant extract-based diets in a 60-day experiment ................................................................................. 91 Table 4.1 Experimental feed ingredients and formulation ......................................... 100 Table 4.2 RBCs ( 106 cells/mm3) of P. hypophthalmus fingerlings under various temperatures in ........................................................................................... 105 Table 4.3 Hb (mg/100 mL) of P. hypophthalmus fingerlings under various temperatures in 14 days .............................................................................. 106 Table 4.4 Hct (%) of P. hypophthalmus fingerlings under various temperatures in 14 days ....................................................................................................... 107 Table 4.5 MCV (µm3) of P. hypophthalmus fingerlings under various temperatures in 14 days ................................................................................................... 107 Table 4.6 MCH (pg) of P. hypophthalmus fingerlings under various temperatures in 14 days ................................................................................................... 108 xi Table 4.7 MCHC (%) of P. hypophthalmus fingerlings under various temperatures in 14 days ................................................................................................... 108 Table 4.8 Glucose concentration (mg/100 mL) of P. hypophthalmus fingerlings under various temperatures in 14 days ....................................................... 109 Table 5.1 Pepsin of P. hypophthalmus fingerlings exposed to different salinities for 42 days. ................................................................................................. 134 Table 5.2 Gastric amylase of P. hypophthalmus fingerlings exposed to different salinities for 42 days. ................................................................................. 134 Table 5.4 Chymotrypsin of P. hypophthalmus fingerlings exposed to different salinities for 42 days. ................................................................................. 136 Table 6.1 Glucose concentration (mg/100 mL) of P. hypophthalmus fingerlings exposed to different nitrite concentrations for 14 days 133 Table 6.2 LPO-gill (nmol MDA/g) of P. hypophthalmus fingerlings exposed to different nitrite concentrations for 42 days ................................................ 138 Table 6.3 LPO-brain (nmol MDA/g) of P. hypophthalmus exposed to different nitrite concentrations for 42 days ............................................................... 139 Table 6.4 LPO-muscle (nmol MDA/g) of P. hypophthalmus exposed to different nitrite concentrations for 42 days ............................................................... 139 Table 6.5 CAT-liver (U/min/mg protein) of P. hypophthalmus exposed to different nitrite concentrations for 42 days ............................................................... 140 Table 6.6 CAT-brain (U/min/mg protein) of P. hypophthalmus exposed to different nitrite concentrations for 42 days ............................................................... 141 Table 6.7 CAT-muscle (U/min/mg protein) of P. hypophthalmus exposed to different nitrite concentrations for 42 days ............................................................... 141 Table 7.1 Physiological parameters of striped catfish fed extract-based diets at 31°C and 35°C compared to 27°C ......................................................... 151 Table 7.2 Physiological parameters of striped catfish fed extract-based diets at 10‰ and 20‰ compared to 0‰ ........................................................... 152 Table 7.3 Physiological parameters of striped catfish fed extract-based diets at 0.08 mM and 0.8 mM compared 0 mM ..................................................... 154 xii LIST OF ABBREVIATION CAT Catalase DWG Daily weight gain FAO Food and Agriculture Organization FCR Feed conversion ratio Fig. Figure GW Gained weight Hb Haemoglobin Hct Haematocrit LC50 96 h Lethal concentration in 96 hours LPO Lipid peroxidation MCH Mean corpuscular haemoglobin MCHC Mean corpuscular haemoglobin concentration MCV Mean corpuscular volume MRD Mekong River Delta Mt Metric ton MIC Minimum inhibitory concentration - NO2 Nitrite RBCs Red blood cells SEM Standard error of mean SGR Specific growth rate SR Survival rate WG Weight gain WBCs Number of white blood cells (leukocytes) Ai Azadirachta indica Eh Euphorbia hirta Mp Mimosa pudica Pa Phyllanthus amarus Pg Psidium guajava xiii ABSTRACT Striped catfish (Pangasianodon hypophthalmus) is an important species of aquaculture in the Mekong River Delta (MRD) of Vietnam. However, it has been constrained by several obstacles, among them which are climate change and diseases. Plant extracts as a dietary supplement is regarded as the easiest and most efficient strategy to improve antioxidant activity while contributing to the stress mitigation. The study aimed to evaluate the effect of selected plant extracts medicated in feed on physiological haematological parameters, digestive enzyme activities and growth and stress responses of striped catfish exposed to environmental stressors (salinity, - temperature and NO2 ). The final aim would be a selection of plant extracts that have a positive effect on fish to reduce the use of antibiotics and avoid water environmental pollution. This doctoral dissertation was, therefore structured into four separate experiments. First, five plant extracts, 0.4% or 2% Euphorbia hirta (Eh), 0.2% or 1% Phyllanthus amarus (Pa), 0.4% or 2% Mimosa pudica (Mp), 0.2% or 1% Psidium guajava (Pg), and 0.4% or 2% Azadirachta indica (Ai), were investigated on haematology, digestive enzyme activities and growth throughout the duration of 60 days. These extracts were identified based on the promising and applicable findings regarding the immunity and antioxidant capacity of striped catfish reported (Nhu et al., 2019; Dao et al., 2020). P. hypophthalmus fingerlings' haematological indices and digestive enzyme activities were modified after sixty days of oral administration with Pg 0.2% or Pa 0.2% extracts, resulting in improved growth performance. Second, the effects of Psidium guajava L. (0.2%) - Pg0.2 and Phyllanthus amarus (0.5%) – Pa0.5 on haematology, thermal stress tolerance, enzymatic activities, and growth of striped catfish subjected to temperatures of 27°C, 31°C, and 35°C for 42 days were examined. Although haematological indicators were most significant at 35°C, they were not significantly different from results noted at 31°C on day 14 post-temperature challenge. The glucose concentration elevated on the third post-temperature challenge day subsequently decreased and remained constant at 35°C until the end of the trial, which was not significantly different compared to those at 27°C. After 42 days, the Pg0.2 and mix diets substantially lowered lipid peroxidation and increased catalase in the gills and liver. Digestive enzymes (trypsin, chymotrypsin, amylases, and pepsin) were accelerated by the Pg0.2 and mix treatments, and enzymatic activity improved from 31°C to 35°C. Overall, fish maintained at 31°C presented the most favorable growth performance, followed by those reared at 35°C, and there was no significant difference in survival rates among these treatments. Assuming the Mekong Delta's average water temperature remains below 35°C, feeding diets incorporating Pg0.2 or Mix (Pg0.2+Pa0.5) extracts strengthen fish health via haematology and oxidative stress resistance. xiv Third, the haematology, digestion, oxidative stress, and growth of striped catfish subjected to three salinity levels (0, 10, and 20‰) in formulated extract-based diets (P. guajava, P. amarus, and a mixture of these two extracs) were investigated. The haematological parameters recovered after three days of exposure to 10‰ and were stable for 14 days; however, this was not observed in 20‰. At 0-10‰, higher digestive enzyme activities (trypsin, chymotrypsin, amylase, and pepsin) was noticed, while a substantial reduction was observed at 20‰ in extended exposures (day 42). On day 42 of high salinity exposure, LPO levels in muscle, liver, brain, and gills were considerably higher at 20‰ than lower tested salinities. Fish can thrive normally up to a salinity of 10‰, and no serious damage to fish organs was identified throughout all salinity levels and sampling durations. This preliminary study revealed that the striped catfish responded more strongly to increased salinity exposure, suggesting that this species might serve as a model bio-indicator in coastal farming. Fourth, fish was susceptible to nitrite exposure and reveal alterations to biological indicators and stress response when randomly administered to the aforementioned extract-based diets for 42 days at (0, 0.08, and 0.8 mM) nitrite concentrations. After 24 hours of exposure to 0.8 mM nitrite, the acquired haematological markers decreased substantially. Almost all digestive enzyme activities in fish exposed to 0.8 mM nitrite decreased from 7 to 42 days after exposure. Nitrite concentration at 0.8 mM resulted in alterations that impaired the antioxidant system (reduced CAT activity) and enhanced oxidative damage in lipids (LPO). Meanwhile, as a result of the high nitrite concentration, the gills were the most severely injured organs. Eventually, 0.8 mM nitrite concentrations damaged the antioxidant system and produced stress in striped catfish fingerlings. The study's findings convey a scientific foundation for assessing the effects of climate change on the aquaculture sector in general, and striped catfish farming in particular. The findings of the study also assist farmers to comprehend the positive and negative impacts of temperature, salinity, and nitrite on striped catfish, simultaneously, assisting farmers to incorporate the positive impacts of preserving environmental elements at optimal levels to promote metabolic activities, as well as beneficial effects on growth to enhance farming production. Furthermore, farmers can augment the dietary intake of striped catfish fingerlings with essential antioxidant components found in plant extracts such as P. guajava 0.2% or combine P. amarus 0.5% to improve health, contribute to the growth and mitigate stress under the current climate change scenario. xv TÓM TẮT Cá tra (Pangasianodon hypophthalmus) là đối tượng nuôi quan trọng của vùng Đồng bằng sông Cửu Long (ĐBSCL), Việt Nam. Tuy nhiên, ngành nuôi cá tra hiện này đang đối mặt với một số trở ngại, trong đó có thể kể đến là biến đổi khí hậu và dịch bệnh trên cá nuôi. Chất chiết thực vật hiện nay được bổ sung vào chế độ ăn của cá tra được đánh giá là phương pháp dễ dàng và hiệu quả nhất để cải thiện hoạt động chống oxy hóa đồng thời góp phần giảm stress trên cá nuôi. Nghiên cứu được thực hiện nhằm đánh giá ảnh hưởng của một số chiết xuất thực vật được bổ sung vào chế độ ăn của cá tra thông qua một số chỉ tiêu sinh lý máu, hoạt tính của enzyme tiêu hóa, tăng trưởng và giảm stress trên cá tra khi tiếp xúc với các tác nhân gây stress từ môi trường (độ mặn, nhiệt độ và nitrite). Mục tiêu chính yếu nhằm sàng lọc các chất chiết thực vật có tác động tích cực đến sức khỏe cá tra nuôi để hạn chế tối đa việc sử dụng kháng sinh và tránh ô nhiễm môi trường nuôi. Luận án tiến sĩ này bao gồm thành bốn thí nghiệm được thực hiện riêng biệt. Thí nghiệm 1, năm loại chiết xuất thực vật khác nhau bao gồm cỏ sữa (Euphorbia hirta) (Eh) 0,4%, 2%; diệp hạ châu (Phyllanthus amarus) (Pa) 0,2%, 1%; mắc cỡ (Mimosa pudica) (Mp) 0,4%; ổi (Psidium guajava) (Pg) 0,2%, 1%, và sầu đâu (Azadirachta indica) (Ai) 0,4%; 2%, đã được nghiên cứu về sinh lý máu, hoạt tính của enzyme tiêu hóa và tăng trưởng trên cá tra trong 60 ngày. Các chất chiết thực vật này được chọn dựa trên những kết quả nghiên cứu trước đó về khả năng miễn dịch và chống oxy hóa tốt trên cá tra (Nhu et al., 2019; Dao et al., 2020). Các chỉ tiêu sinh lý máu và hoạt tính của enzyme tiêu hóa của cá tra được cải thiện sau 60 ngày bổ sung chất chiết Pg 0,2% hoặc Pa 0,2% vào chế độ ăn, dẫn đến tăng trưởng được cải thiện. Thí nghiệm 2, ảnh hưởng của chất chiết ổi (0,2%) – Pg0,2 và diệp hạ châu (0,5%) – Pa0,5 qua các chỉ tiêu sinh lý máu, khả năng đáp ứng stress, hoạt tính enzyme tiêu hóa và tăng trưởng của cá tra ở nhiệt độ 27°C, 31°C và 35°C trong 42 ngày đã được thực hiện. Mặc dù các chỉ tiêu sinh lý máu thay đổi có ý nghĩa thống kê ở 35°C, nhưng khác biệt không có ý nghĩa thống kê so với kết quả được ghi nhận ở 31°C sau 14 ngày dưới tác động của nhiệt độ. Nồng độ glucose tăng cao vào ngày thứ ba dưới ảnh hưởng của nhiệt độ sau đó giảm xuống và duy trì nồng độ glucose ở 35°C cho đến khi kết thúc thí nghiệm, khác biệt không đáng kể so với nồng độ ở 27°C. Sau 42 ngày, chế độ ăn có bổ sung Pg0,2 và hỗn hợp chất chiết (Pg0,2+Pa0,5) làm giảm đáng kể quá trình oxy hóa lipid và tăng catalase trong mang và gan. Các enzyme tiêu hóa (trypsin, chymotrypsin, amylase và pepsin) được tăng cường ở chế độ ăn có bổ sung Pg0,2 và hỗn hợp, đồng thời hoạt tính của enzyme được cải thiện ở nhiệt độ từ 31°C đến 35°C. Nhìn chung, cá nuôi ở nhiệt độ 31°C có tăng trưởng tốt nhất nhất, kế đế đến là ở nhiệt độ 35°C và khác biệt không có ý nghĩa thống kê về tỷ lệ sống giữa hai mức nhiệt độ này. Nếu nhiệt độ trung bình của ĐBSCL dưới 35°C, và chế độ ăn có bổ sung Pg0,2 hoặc hỗn hợp xvi (Pg0,2+Pa0,5) giúp tăng cường sức khỏe của cá thông qua các chỉ tiêu sinh lý máu và khả năng chống stress oxy hóa ở cá. Thí nghiệm 3, các chỉ tiêu sinh lý máu, enzyme tiêu hóa, oxy hóa stress và tăng trưởng của cá tra với mức độ mặn (0, 10 và 20 ‰) và chế độ ăn có bổ sung chất chiết từ ổi, diệp hạ châu và hỗn hợp của hai chất chiết này) đã được thực hiện. Các chỉ tiêu sinh lý máu phục hồi sau ba ngày tiếp xúc với độ mặn 10‰ và duy trì trong 14 ngày; tuy nhiên, điều này không được ghi nhận ở độ mặn 20‰. Ở khoảng độ mặn 0-10‰, hoạt tính của enzyme tiêu hóa (trypsin, chymotrypsin, amylase và pepsin) cao hơn, trong khi đó, hoạt tính enzyme tiêu hóa giảm đáng kể ở độ mặn 20‰ khi tiếp xúc trong thời gian dài (ngày 42). Vào ngày thứ 42 tiếp xúc với độ mặn cao, nồng độ LPO trong cơ, gan, não và mang của cá tra cao hơn ở độ mặn 20‰ so với độ mặn thấp hơn. Cá có thể phát triển bình thường ở độ mặn 10‰ và các cơ quan của cá không bị ảnh hưởng đáng kể trong tất cả thời gian lấy mẫu. Nghiên cứu sơ bộ này cho thấy rằng cá tra có ảnh hưởng càng lớn khi tiếp xúc với độ mặn tăng lên quá cao (20%), điều này cho thấy rằng loài này có thực hiện nuôi ở các vùng nước lợ ven biển. Thứ nghiệm 4, cá tra được tiếp xúc với các nồng độ nitrite khác nhau và thể hiện những thay đổi thông qua các chỉ tiêu sinh lý máu và khả năng đáp ứng stress khi được bổ sung vào chế độ ăn các loại chất chiết xuất đã đề cặp ở các thí nghiệm trên trong 42 ngày với các nồng độ nitrite khác nhau (0; 0,08 và 0,8 mM). Sau 24 giờ tiếp xúc với nitrite ở nồng độ 0,8 mM, các chỉ tiêu sinh lý máu đã giảm đáng kể. Hầu như tất cả hoạt tính của enzyme tiêu hóa ở cá tiếp xúc với 0,8 mM nitrite đều giảm từ giai đoạn 7 đến 42 ngày sau khi tiếp xúc với. Nồng độ nitrite 0,8 mM gây ra những thay đổi làm suy yếu hệ thống chống oxy hóa (giảm hoạt tính CAT) và tăng cường oxy hóa lipid (LPO). Trong khi đó, do nồng độ nitrite cao, mang là cơ quan bị ảnh hưởng nặng nề nhất. Chính vì vậy, nồng độ nitrite 0,8 mM đã ảnh hưởng đáng kể đến hệ thống chống oxy hóa và gây ra stress trên cá tra giai đoạn giống. Tóm lại, kết quả của nghiên cứu là cơ sở khoa học góp phần đánh giá ảnh hưởng của điều kiện biến đổi khí hậu lên ngành nuôi trồng thủy sản nói chung và nghề nuôi cá tra nói riêng. Kết quả nghiên cứu cũng giúp người nuôi nhận biết được ảnh hưởng có lợi và bất lợi của nhiệt độ, độ mặn và nitrite lên cá tra. Đồng thời, giúp người nuôi ứng dụng ảnh hưởng tốt của việc duy trì các yếu tố môi trường ở mức thích hợp nhằm tăng cường hoạt động trao đổi chất, ảnh hưởng tốt đến tăng trưởng có thể tăng năng suất nuôi. Bên cạnh đó, nhờ vào các thành phần chống oxy hóa hữu ích có trong chất chiết thực vật như ổi và diệp hạ châu, người nuôi có thể ứng dụng bổ sung vào chế độ ăn của cá tra giai đoạn giống nhằm tăng cường sức khỏe và giảm stress, góp phần vào tăng trưởng cho cá nuôi trong điều kiện biến đổi khí hậu hiện nay. xvii CHAPTER 1 INTRODUCTION 1.1 General introduction The development of striped catfish (Pangasianodon hypophthalmus) aquaculture in particular for the Mekong River Delta (MRD), Viet Nam has been challenged with various issues, of which climate change and disease are the most concern of the fish farmers and researchers. This region is most likely to be affected by global climate change. In years ago, the average temperature in Viet Nam increased approximately 0.5- 0.7°C; and the annual average temperature of Viet Nam will increase by 2.3°C by the end of the 21st century (MONRE, 2009); this will greatly affect the sustainable development not only fisheries but also aquaculture including striped catfish. As fish is poikilothermic species, therefore temperature is the main factor that cause affects directly or indirectly to fish life. Changing temperature can be negative effects to fish by increasing or reducing metabolic rates (Galloway and Kieffer, 2003), swimming performance (Hocutt, 1973), impairing immune functions (Hurst, 2007), consequence in reducing the ability to capture prey, increasing susceptibility to disease and enhancing mortality (Donaldson et al., 2008). Temperature shock can also impede predator avoidance (Ward and Bonar, 2003), alter rates of recovery from exercise (Suski et al., 2006), and disrupt homeostasis (Vanlandeghem et al., 2010). The MRD is also one of three extensive low topographical regions in Viet Nam that have been predicted to be impacted by sea-level rises this century (Parry et al., 2007). If sea-level rise occurs as predicted, the striped catfish industry in Viet Nam is likely to be severely impacted because the region is flat and low lying with a maximum elevation of less than 4.0 m above mean sea level. If predictions of future climate change of this century are realized, a 1m increase in mean sea level will result in approximately 1,000 km2 of cultivated land and farming area in Viet Nam becoming salt marshland, and 15,000 - 20,000 km2 of the MRD is likely to be flooded by seawater (i.e. up to 30‰) (Parry et al., 2007). Increasing salinization of freshwater areas will, as a consequence, impact the large local freshwater aquaculture industry, in particular the striped catfish culture, as a result of changes to both soil and local freshwater resources (Sebesvari et al., 2011). In addition, intensive ponds with overfeeding and wastes may lead to the decomposition processes of organic matters. Toxic gases are significantly generated in hypoxic and high-temperature conditions such as ammonia, nitrite, nitrate, carbon dioxide and hydrogen sulfite. Especially, nitrite is a naturally occurring component of the nitrogen cycle, which under some circumstances can reach high levels where it can present a problem, because of well-documented toxicity to animals (Lewis and Morris, 1986), typically for freshwater species. Methaemoglobin formation from reaction of nitrite with haemoglobin (Hb) is the main reason for the exceptional low arterial O2, but 18